1. Field of the Invention
The present invention relates to a protein refolding method, in particular, relates to the method of protein refolding with ion exchange resins and the application of the same. The invention belongs to the technical field of protein refolding in biotechnology.
2. Description of the Related Art
Since the advent of recombinant DNA technology in the early 1970s, the rapid development of biotechnology has greatly propelled the mass production of recombinant proteins. To produce a recombinant protein, Escherichia coli (E. coli) is usually the preferred choice as the host because it has many advantages, such as simple plasmid construct, low nutrient requirement, fast growth, high level of expression, and ease of separation. Many of the over-expressed target proteins accumulate as inclusion body in E. coli. The inclusion body has correct primary structure but no high-level structure. Thus, it has no biological activity and has to be refolded to achieve its native bioactivity. Because of the complicated protein structure, refolding is a difficult technology during the production of biological pharmaceutical proteins in the gene project. A major challenge in protein refolding is the aggregation of folding intermediates, which is parallel to the on-pathway folding method and is the leading cause of low refolding yield. Therefore, the inhibition of the aggregation is the great challenge for protein refolding in industrial production.
One strategy to inhibit the formation of the aggregation is to use folding additives in the refolding system. However, additive-assisted methods bring the trouble of further separation due to the presence of additives, and most of folding additives are not reusable. Moreover, protein chaperones are too expensive for industrial applications.
On-column refolding by chromatography is another approach to inhibiting aggregation of folding intermediates. But there is no report about the method for protein refolding with ion exchange resins as an additive. Protein refolding by chromatography is usually carried out in two modes, flow-through mode (size exclusion chromatography) and adsorptive mode (hydrophobic interaction chromatography, ion-exchange chromatography, and affinity chromatography). Unfolded proteins are adsorbed in the chromatographic columns by hydrophobic interactions, electrostatic interactions, or specific affinity and thus hydrophobic interaction between protein molecules can effectively be inhibited. However, chromatography is low throughput, facilities of chromatography are rather expensive and need high-cost maintenance, columns are prone to blockage by protein aggregation, high-quality degassed and filtrated mobile phases are needed, and operation is relatively complex. In addition to these problems, it is usually difficult to achieve refolding products with high concentrations by the on-column method. So far, no application of ion exchange resins with the same sign of charge as the proteins to be refolded has been reported for protein refolding.
To overcome the drawbacks of refolding with additives and chromatography, the present invention provides a method for protein refolding with the ion exchange resins as an additive. The method provided has several advantages over other methods for protein refolding including no requirement for chromatographic facilities, simple operation and no additional contaminant introduced during the process of refolding. Finally, the ion exchange resins can be recycled via solid-liquid separation and regeneration.